Hogg S. Essential microbiology

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128 MICROBIAL METABOLISM

Aerobic respiration

We shall now examine the fate of the pyruvate produced as the end-product of glycol-

ysis. As we have seen, this depends on whether the organism in question is aerobic or

anaerobic.

You will recall that during glycolysis, NAD

was reduced to NADH. In order for glu-

cose metabolism to continue, this supply of NAD

must be replenished; this is achieved

either by respiration or fermentation. Respiration is the term used to describe those ATP-

generating processes, aerobic or anaerobic, by which oxidation of a substrate occurs,

with an inorganic substance acting as the ﬁnal electron acceptor.Inaerobic respira-

tion, that substance is oxygen; in anaerobic respiration, a substance such as nitrate or

sulphate can fulﬁl the role.

The TCA cycle is a series

of reactions that oxidize

acetate to CO

, gener-

ating reducing power in

the form of NADH and

FADH

for use in the elec-

tron transport chain.

In most aerobic organisms, the pyruvate is completely

oxidised to CO

and water by entering the tricarboxylic

acid (TCA) cycle, also known as the Krebs cycle or sim-

ply the citric acid cycle (Figure 6.20). During this cycle,

a series of redox reactions result in the gradual transfer

of the energy contained in the pyruvate to coenzymes

(mostly NADH). This energy is ﬁnally conserved in the

form of ATP by a process of oxidative phosphorylation.

We shall turn our attention to these important reactions

in due course, but ﬁrst let us examine the role of the TCA cycle in a little more detail.

Pyruvate does not itself directly participate in the TCA cycle, but must ﬁrst be con-

verted into the two-carbon compound acetyl-Coenzyme A:

Pyruvate Acetyl-CoA + CO

(2C)

NAD

NADH

+ H

(3C)

Coenzyme A

This is an important intermediate, as lipids and amino acids can also be metabolised

into this form, and thereby feed into the TCA cycle. The main features of the cycle are

as follows:

each reaction is catalysed by a separate enzyme

four of the reactions involve substrate oxidation, with energy, in the form of

electrons, passing to form NADH (mainly) and FADH

the two carbons present in acetyl-CoA are removed as CO

one reaction involves the generation of ATP by substrate-level phosphorylation.

For each ‘turn’ of the citric acid cycle, one molecule of ATP, three molecules of NADH

and one molecule of FADH

are produced (FADH

is the reduced form of another

coenzyme, FAD). Since these derive from oxidation of a single acetyl-CoA molecule,

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PRINCIPLES OF ENERGY GENERATION 129

Figure 6.20 The TCA cycle. Acetyl-CoA may derive from the pyruvate of glycolysis or

from lipid or amino acid metabolism. It joins with the four-carbon oxaloacetate to form the

six-carbon citric acid. Two decarboxylation steps reduce the carbon number back to four and

oxaloacetate re-enters the cycle once more. Although no ATP results directly from the cycle,

the third phosphate on GTP can be easily transferred to ADP (GTP + ADP = GDP + ATP),

thus generating one molecule of ATP per cycle. In addition, substantial reducing power is

generated in the form of NADH and FADH

. These carry electrons to the electron transport

chain, where further ATPs are generated

we need to double these values per molecule of glucose originally entering glycolysis.

Several of the intermediate molecules in the TCA cycle also act as precursors in other

pathways, such as the synthesis of amino acids, fatty acids or purines and pyrimidines

(see Anabolic metabolism, below). Other pathways regenerate such intermediates for

continued use in the TCA cycle (see Box 6.3).

So far, we are a long way short of the 38 molecules of ATP per molecule of glucose

mentioned earlier; we have only managed two ATPs from glycolysis and a further two

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130 MICROBIAL METABOLISM

Box 6.3 The glyoxylate cycle

The components of the TCA cycle may act as precursors for the biosynthesis of other

molecules (e.g., both α-ketoglutarate and oxaloacetate can be used for the synthe-

sis of amino acids). For the TCA cycle to continue, it must replace these compounds.

Many microorganisms are able to do this by converting pyruvate to oxaloacetate

via a carboxylation reaction. A pathway that replenishes intermediate compounds

of another in this way is termed anaplerotic. Organisms that use acetate (or molecules

that give rise to it e.g. fatty acids) as sole carbon source regenerate TCA interme-

diates by means of the glyoxylate cycle (sometimes known as the glyoxylate shunt or

bypass). This resembles the TCA cycle, but the two decarboxylation reactions (i.e.

those where CO

is removed) are missed out (compare with Figure 6.20).

Succinate

α-Ketaglutarate

Isocitrate

Citrate

Fumarate

Malate

Oxaloacetate

SuccinylCoA

AcetylCoA CoA

Fatty Acids

CoA Acetyl

CoA

Malate

synthase

Isocitrate

Iyase

Glyoxylate

CHCO

–

NADH

NAD

Thus, isocitrate is converted directly to succinate and glyoxylate, and in another

unique reaction, the glyoxylate is joined by acetyl-coA to form malate. The result of

this is that succinate can be removed to participate in a biosynthetic pathway, but

oxaloacetate is still renewed via glyoxylate and malate.

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PRINCIPLES OF ENERGY GENERATION 131

NADH

NAD

FADH

FM N FAD

Cyt b

Energy Cyt c

Cyt c

Cyt a

Figure 6.21 The electron transport chain. Electrons from NADH and FADH

pass from

one electron carrier to another, with a gradual release of energy as ATP by chemiosmosis (see

Figure 6.22). The electron carriers are arranged in order of their reduction potential (tendency

to gain electrons) and oscillate between the oxidised and the reduced state. FMN = ﬂavin

mononucleotide, Q = coenzyne Q.

from the TCA cycle. Where do all the rest come from? Most of the energy originally

stored in the glucose molecule is now held in the form of the reduced coenzymes (NADH

and FADH

) produced during glycolysis and the TCA cycle. This is now converted to

no less than 34 molecules of ATP per glucose molecule by oxidative phosphorylation

in the remaining steps in aerobic respiration (three from each molecule of NADH and

two from each of FADH

The electron transport

chain is a series of donor/

acceptor molecules

that transfer electrons

from donors (e.g. NADH)

to a terminal electron

acceptor (e.g. O2).

In the ﬁnal phase of aerobic respiration, electrons

are transferred from NADH and FADH

, via a series

of carrier molecules known collectively as the electron

transport (or respiratory) chain to oxygen, the terminal

electron acceptor (Figure 6.21). This in turn is reduced

to the molecules of water you will remember from our

overall equation on page 122. In procaryotes, this elec-

tron transfer occurs at the plasma membrane, while in

eucaryotes it takes place on the inner membrane of mi-

tochondria. Table 6.2 summarises the locations of the reactions in the different phases

of carbohydrate metabolism.

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132 MICROBIAL METABOLISM

Table 6.2 Location of respiratory enzymes

Reaction Procaryotes Eucaryotes

Glycolysis Cytoplasm Cytoplasm

TCA cycle Cytoplasm Mitochondrial matrix

Electron Transport Plasma membrane Mitochondrial inner

membrane

Oxidative phosphorylation and the electron transport chain

The components of the electron transport chain differ between procaryotes and eu-

caryotes, and even among bacterial systems, thus details may differ from the example

outlined below. The purpose of the electron transport is the same for all systems, how-

ever, that is, the transfer of electrons from NADH/FADH

via a series of carriers to,

ultimately, oxygen. Around half of the energy released during this process is conserved

as ATP.

The carrier molecules, which act alternately as acceptors and donors of electrons,

are mostly complex modiﬁed proteins such as ﬂavoproteins and cytochromes, together

with a class of lipid-soluble molecules called ubiquinones (also called coenzyme Q). The

carriers are arranged in the chain such that each one has a more positive redox potential

than the previous one. In the ﬁrst step in the chain, NADH passes electrons to ﬂavin

mononucleotide (FMN), and in so doing becomes converted back to NAD

, thereby

ensuring a ready supply of the latter for the continuation of glycolysis (Figure 6.21).

From FMN, the electrons are transferred to coenzyme Q, and thence to a series of

cytochromes; at each transfer of electrons the donor reverts back to its oxidised form,

ready to pick up more electrons. You may recall that FADH

yields only two, rather than

three molecules of ATP per molecule; this is because it enters the electron transport chain

at a later point than NADH, thereby missing one of the points where export of protons

occurs. The ﬁnal cytochrome in the chain transfers its electrons to molecular oxygen,

which, as we have seen, acts as the terminal oxygen acceptor. The negatively charged

oxygen combines with protons from its surroundings to form water. Four electrons and

protons are required for the formation of each water molecule:

+ 4e

−

+ 4H

−→ 2H

Since two electrons are released by the oxidation of each NADH, it follows that two

NADH are needed for the oxidation of each oxygen.

How does this transfer of electrons lead to the formation of ATP? The chemiosmotic

theory proposed by Peter Mitchell in 1961 offers an explanation. Although it was not

immediately accepted, the validity of the chemiosmotic model is now widely recognised,

and in 1978 Mitchell received a Nobel Prize for his work. As envisaged by Mitchell,

sufﬁcient energy is released at three points in the electron transport chain for the transfer

of protons to the outside of the membrane, resulting in a gradient of both concentration

and charge (proton motive force). The protons are able to return across the membrane

and achieve an equilibrium through speciﬁc protein channels within the enzyme ATP

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PRINCIPLES OF ENERGY GENERATION 133

Figure 6.22 Chemiosmosis. The active transport of protons across the membrane creates

a gradient of charge and concentration (proton motive force). Special channels containing

ATP synthase allow the return of the protons; the energy released is captured as ATP. From

Hames, BD, Hooper, NM & Houghton, JD: Instant Notes in Biochemistry, Bios Scientiﬁc

Publishers, 1997. Reproduced by permission of Thomson Publishing Services

synthase. The energy released by the protons as they return through these channels

enables the ATP synthase to convert ADP to ATP (Figure 6.22).

Aerobic respiration in eucaryotes is slightly less efﬁcient than in procaryotes due to

the fact that the three stages take place at separate locations (see Table 6.2). Thus the

total number of ATPs generated is 36 rather than the 38 in procaryotes (Table 6.3).

Fermentation

We turn now to the fate of pyruvate when oxygen is not available for aerobic respira-

tion to take place. Microorganisms are able, by means of fermentation, to oxidise the

pyruvate incompletely to a variety of end products.

Table 6.3 Yield of ATP by aerobic respiration in procaryotes

Process ATP Yield (per glucose molecule)

Glycolysis 2

TCA Cycle 2

Electron transport chain 34

∗

Derived from the oxidative phosphorylation of 2 × NADH from glycolysis,

8 × NADH and 2 × FADH

from TCA cycle.

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134 MICROBIAL METABOLISM

It is worth spending a moment deﬁning the word fermentation. The term can cause

some confusion to students, as it has come to have different meanings in different

contexts. In everyday parlance, it is understood to mean simply alcohol production,

while in an industrial context it generally means any large scale microbial process such as

beer or antibiotic production, which may be aerobic or anaerobic. To the microbiologist,

the meaning is more precise:

a microbial process by which an organic substrate (usually a carbohydrate) is broken down

without the involvement of oxygen or an electron transport chain, generating energy by substrate-

level phosphorylation

Two common fermentation pathways result in the production of respectively lactic

acid and ethanol. Both are extremely important in the food and drink industries, and

are discussed in more detail in Chapter 17. Alcoholic fermentation, which is more

common in yeasts than in bacteria, results in pyruvate being oxidised via the intermediate

compound acetaldehyde to ethanol.

COCOO

–

CHO CH

Pyruvate Acetaldehyde Ethanol

pyruvate decarboxylase alcohol dehydrogenase

NAD

NADH

Electrons pass from the reduced coenzyme NADH to acetaldehyde, which acts as an

electron acceptor, and NAD

is thereby regenerated for use in the glycolytic pathway.

No further ATP is generated during these reactions, so the only ATP generated in the

fermentation of a molecule such as glucose is that produced by the glycolysis steps. Thus,

in contrast to aerobic respiration, which generates 38 molecules of ATP per molecule

of glucose, fermentation is a very inefﬁcient process, producing just two. Note that all

the ATP produced by any fermentation is due to substrate-level phosphorylation, and

does not involve an electron transport chain.

A variety of microorganisms carry out lactic acid fermentation. Some, such as Strep-

tococcus and Lactobacillus, produce lactic acid as the only end product; this is referred

to as homolactic fermentation.

COCOO

–

CHOHCOO

–

Pyruvate Lactate dehydrogenase

NAD

NADH

Lactate

Certain other microorganisms, such as Leuconostoc, generate additional products such

as alcohols and acids in a process called heterolactic fermentation.

In both alcohol and lactic acid fermentation, the two NADH molecules produced

per molecule of glucose have been reoxidised to NAD

, ready to re-enter the glycolytic

pathway.

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PRINCIPLES OF ENERGY GENERATION 135

Figure 6.23 Fermentation patterns in enteric proteobacteria. All members of the group

produce pyruvate via the Embden–Meyerhof pathway (glycolysis), but subsequent reactions

fall into one of two main types. (a) Mixed acid fermentation results in ethanol and a mixture

of acids, mainly acetic, lactic, succinic and formic, e.g. Escherichia, Salmonella. (b) Butane-

diol fermentation involves the conversion of pyruvate to acetoin, then to 2,3-butanediol, e.g.

Enterobacter, Klebsiella. The ratio of CO

to H

production is much greater in butanediol

fermentation

Other types of fermentation

Members of the enteric bacteria metabolise pyruvate to a variety of organic compounds;

two principal pathways are involved, both of them involving formic acid. In mixed acid

fermentation, pyruvate is reduced by the NADH to give succinic, formic and acetic acids,

together with ethanol (Figure 6.23a). Escherichia, Shigella and Salmonella belong to

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136 MICROBIAL METABOLISM

this group (see Chapter 7). Other enteric bacteria, such as Klebsiella and Enterobacter,

carry out 2,3-butanediol fermentation. In this, the products are not acidic, and include

an intermediate called acetoin. Much more CO

is produced per molecule of glucose

than in mixed acid fermentation (Figure 6.23b). The end products of the two types of

fermentation provide a useful diagnostic test for the identiﬁcation of unknown enteric

bacteria.

Metabolism of lipids and proteins

We have concentrated so far on the metabolism of carbohydrates, but both lipids and

proteins may also act as energy sources. Both are converted by a series of reactions to

an intermediate compound that can then enter the pathways of metabolism we have

discussed above.

Lipids often form an important energy source for microorganisms; they are plentiful

in nature, as they form the major component of cell membranes, and may also exist as

cellular storage structures. Lipids are hydrolysed by a class of enzymes called lipases to

their constituent parts; the fatty acids so produced enter the cyclic β-oxidation pathway.

In this, fatty acids are joined to coenzyme A to form an acyl-CoA, and shortened by two

carbons in a series of reactions (Figure 6.24). Molecules of NADH and FADH

derived

from β-oxidation can enter the electron transport chain to produce ATP. Acetyl-CoA,

you will recall from earlier in this chapter, serves as the entry point into the TCA cycle.

When you consider that a single turn of the TCA cycle gives rise to the production of 14

molecules of ATP, you can appreciate what a rich source of energy a 16- or 18-carbon

fatty acid represents. The glycerol component of a lipid requires only slight modiﬁcation

in order to enter the glycolytic pathway as dihydroxyacetone phosphate (see ‘Glycolysis’

above).

Proteins are a less useful source of energy than lipids or carbohydrates, but may be

utilised when these are in short supply. Like lipids, they are initially hydrolysed to their

constituent ‘building blocks’, in this case, amino acids. These then undergo the loss of

an amino group (deamination), resulting in a compound that is able to enter, either

directly or indirectly, the TCA cycle.

CCH COOH COO

–

+ NH

Alanine Pyruvate

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→

Figure 6.24 β-oxidation of lipids. β–Oxidation comprises a series of four reactions,

repeated for the removal of each two-carbon unit. Formation of the acyl CoA ester

requires the expenditure of ATP, but there is a net gain in reducing power (NADH + FADH

which can feed into the electron transport chain. The shortened acyl chain at the end of the

process can re-enter the cycle and become further shortened, while acetyl CoA can enter

directly into the TCA cycle (CoA-SH = coenzyme A)

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PRINCIPLES OF ENERGY GENERATION 137

Oxidation

Cleavage

(thiolysis)

Hydration

ATP

AMP

P P 2 P

NAD

NADH + H

FAD

FADH